Spatio-temporal segregation between sensory relay and swallowing pre-motor population activities by optical imaging in the rat nucleus of the solitary tract.

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Shinya Fuse, Yoichiro Sugiyama, Rishi R. Dhingra, Shigeru Hirano, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5104317/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jan, 2025 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted 10 You are reading this latest preprint version Abstract The nucleus tractus solitarius (NTS) contains neurons that relay sensory swallowing commands information from the oropharyngeal cavity and swallowing premotor neurons of the dorsal swallowing group (DSG). However, the spatio-temporal dynamics of the interplay between the sensory relay and the DSG is not well understood. Here we employed fluorescence imaging after microinjection of the calcium indicator into the NTS in an arterially perfused brainstem preparation of rat (n = 8) to investigate neuronal population activity in the NTS in response to superior laryngeal nerve (SLN) stimulation. Respiratory and swallowing motor activities were determined by simultaneous recordings of phrenic and vagal nerve activity (PNA, VNA). Analysis of SLN stimulation near the threshold triggering a swallowing allowed us to analyze Ca 2+ signals related to the sensory relay and the DSG. We show that activation of sensory relay neurons triggers spatially confined Ca 2+ signals exclusively unilateral to the stimulated SLN at short latencies (114.3 ± 94.4 ms). However, SLN-evoked swallowing triggered Ca 2+ signals bilaterally at longer latencies (200 ± 145.2 ms) and engaged anatomically distributed DSG activity across the dorsal medulla oblongata. The Ca 2+ signals originating from the DSG preceded evoked VNA swallow motor bursts, thus the swallowing premotor neurons that drive laryngeal motor pools are located outside the DSG. In conclusion, the study illuminates the spatial-temporal features of sensory-motor integration of swallowing in the NTS and further supports the hypothesis that the NTS harbors swallowing pre-motor neurons that may generate the swallowing motor activity while first order pre-motor pools are located outside the DSG. Swallowing In situ perfused brainstem preparation Optical recording Calcium imaging Dorsal swallowing group Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The pharyngeal phase of swallowing is initiated by a swallowing central pattern generator (sw-CPG) which is traditionally divided into a dorsal and ventral swallowing group (DSG, VSG) [ 1 , 2 , 3 ]. Previous studies characterized the activity of swallowing interneurons (SINs) using single unit recordings predominantly within the DSG [ 4 , 5 ]. SINs are activated by pharyngeal or laryngeal afferent signals [ 3 , 6 , 7 , 8 ]. However, the location of the DSG largely overlaps anatomically with the nucleus of the solitary tract, which also serves as the primary sensory relay nucleus for pharyngeal and laryngeal afferents [ 6 , 9 , 10 ]. The spatio-temporal dynamic of the interplay between the sensory relay and DSG within these circuits of the dorsal medulla oblongata remains to be elucidated. Fluorescence calcium imaging approaches at the population level in slices and en bloc brainstem spinal cord preparation was previously used to analyze temporal and spatial changes in respiratory network activity at either the population or the single cell level [ 11 , 12 , 13 , 14 ]. However, investigation of the network mechanism that underlies the generation of the swallowing motor pattern in relation to breathing requires in vivo experimental models [ 15 , 16 ] or alternative approaches that can maintain network function in an in vitro slice preparation [ 17 ] or in in situ arterially perfused brainstem preparations. The latter has become an established experimental model to study the coordination of breathing and swallowing [ 18 , 19 , 20 , 21 ]. The perfused brainstem preparation was previously used to image respiratory [ 22 ] and swallowing activities [ 23 ] in brainstem circuits and this specific preparation provides several advantages compared to in vivo or in vitro approaches [ 24 ]. The overall mechanical stability in the absence of cardiac pulsation or respiratory movements and the full exposure of the dorsal surface of the brainstem provide a viable experimental approach for calcium imaging and allows for the study of spatio-temporal dynamics of network activity of the DSG and the sensory relay neurons in the dorsal medulla oblongata. In the present study we demonstrate that activation of the sensory inputs triggered a spatially confined population Ca 2+ signal within the NTS at short latency after electrical stimulation of the SLN. In contrast, the generation of the swallowing motor pattern occurred after significantly longer latency and engaged DSG population activities along the rostro-caudal and medio-lateral dorsal medulla. The data of the present study demonstrates for the first time that the sensory relay neurons in the NTS spatially overlap with the DSG but are functionally and anatomically separable. In addition, the present study further supports the hypothesis that swallowing motor activity is generated in the DSG and pre-motor populations in the ventral swallowing group transmit the swallowing network activity to the motor pools [ 3 ] since the Ca 2+ signals of the DSG clearly preceded the onset of swallowing motor burst. Materials and methods The experimental procedures were carried out in accordance with the principles for the Care and Use of Animals of the Physiological Society in Japan and approved by the local University Committee for the Use of Animals in Research, and all experiments were performed at the Hyogo College of Medicine. The in situ arterially perfused-brainstem preparation. Experiments were performed using arterially perfused brainstem preparations as previously described in detail [ 25 ]. A total of eight Sprague-Dawley rats (either sex; postnatal days 15–24; weight: 40.1–43.8 g) were used. Each animal was initially anesthetized with isoflurane. The left phrenic nerve was isolated and cut just above the diaphragm. In the neck, the left vagus nerve and left SLN were isolated and cut distally. The cerebellum was removed by cutting the cerebellar brachium, and the dorsal medulla oblongata was exposed. Artificial cerebrospinal fluid (aCSF; in mM: 125 NaCl, 3 KCl, 1.25 KH2PO4, 2.5 CaCl2, 1.25 MgSO4, 25 NaHCO3, and 10 D-glucose) containing 4.5×10 − 3 g/mL sucrose was perfused through a catheter using a peristaltic pump (Watson and Marlow, 520s). The perfusate was then bubbled continuously with carbogen (95% O2 and 5% CO2). The flow rate was gradually increased to 16–22 ml/min so that the characteristic eupneic post-inspiratory motor discharge was established in the vagal nerve recording [ 26 ]. Neuromuscular paralysis was induced by the addition of vecuronium bromide (initial injection of 1.5 mg/kg, additional injections of 0.75 mg/kg when necessary) to the perfusate which suppressed respiratory-related movements of the preparation. To provide a powerful transient excitatory drive for the respiratory brainstem network during the tuning phase of the preparation peripheral chemoreceptors were occasionally stimulated by injecting sodium cyanide (NaCN; 0.1–0.2 ml, 0.1%, w/v in saline) into the perfusion circuit. Nerve recording In all experiments phrenic nerve activity (PNA) and vagal nerve activity (VNA) were recorded via suction electrodes. The activity of neurons was amplified (AB651J, Nihon Kohden, Tokyo, Japan), bandpass filtered from 15 Hz to 3 kHz, sampled at 800 Hz, and stored together with imaging data using an optical recording system (MiCAM Ultima, BrainVision, Tokyo, Japan). The nerve discharges were high pass filtered (cutoff frequency = 0.1 Hz), fully rectified and leaky integrated at a time constant of 20 ms. Experimental protocol for SLN stimulation For electrical stimulation of the SLN a bipolar electrode was used to apply short trains of electrical pulses [ 20 ] with a duration of 0.2 ms at a frequency of 10–20 Hz and at an intensity of (20–50 µA). Initially we determined the threshold of the stimulus intensity for SLN-evoked swallows which can be reliably identified in vagal nerve recordings [ 20 ]. Subsequently we performed SLN-stimulations at the threshold level during the optical recording sessions (details below). This allowed us to discriminate the optical signals only related to activation of the sensory relay neurons in the NTS in case SLN-stimulation failed to evoke swallowing. SLN-stimulations that succeeded to evoke swallowing motor activity then revealed spatio-temporal network activity of the overlapping DSG. Calcium-sensitive dye microinjection We stained the dorsal medulla with the calcium indicator Oregon Green 488 BAPTA-1 acetoxymethyl (OGB-1, Invitrogen, Carlsbad, CA, USA). OGB-1 was dissolved in 10% Pluronic F-127 in dimethyl sulfoxide (DMSO, Invitrogen, Carlsbad, CA, USA) at a concentration of 200 µM. The microinjection sites were located 0.5–1.0 mm rostral to the obex; 0.5–1.0 mm lateral from the midline on both sides; and 300, 400, 500 µm below the dorsal surface. A total of 50–60 nl of OGB-1 was microinjected into each injection site. Optical recording After dye microinjection, the recording chamber was carefully placed under a macro zoom fluorescence microscope (MVX-10, Olympus Optical, Tokyo, Japan) using a magnification of 1.6 x. (Fig. 1 ). Time-lapsed Ca 2+ signals in the dorsal medulla were imaged using an optical recording system (MiCAM Ultima, BrainVision, Tokyo, Japan). Preparations were illuminated with a tungsten-halogen lamp (150 W) through a bandpass excitation filter (λ = 460–495 nm). Epifluorescence was detected through a long-pass barrier filter (λ > 510 nm) with a CMOS sensor array (MiCAM Ultima L-camera, BrainVision; 100 µm × 100 µm pixel size, 100 × 100 pixel array). Optical signals were sampled at 40 Hz (25 ms/frame). A total of 256 frames were recorded starting at 64 frames (1.6 s) before the onset of SLN electrical stimulation. Image processing First, the change in fluorescence intensity ( ΔF ) relative to the initial intensity ( F0 ) was calculated. Then, the fractional change in fluorescence intensity ( ΔF/F ) at each pixel in each frame was calculated based on the background fluorescence intensity (F). Subsequently, the images were smoothed spatially using a 5 × 5 pixel spatial filter; pre-low-pass image) and temporally using a low-pass filter (cutoff frequency = 2 Hz; low-pass image). Next, we selected 9–20 images from each animal in which SLN stimulation did not elicit swallowing and applied cycle-triggered averaging. For SLN stimulation that elicited swallowing we selected 5–20 images from each animal and also applied cycle-triggered averaging. Finally, the activation map of the low-pass image was drawn using an activation threshold of 2 × SD (standard deviation). The SD was derived from ΔF/F at each pixel within 10 frames before the onset of SLN stimulation in the pre-low-pass image. The activation map for each animal was superimposed based on the bottom of the floor of the fourth ventricle of each animal and merged with a representative normal light-microscope photograph. Data analysis We measured the latency to SLN-elicited swallowing activity at the neurogram and measured the peak and decay times of optical signal changes. The latency of SLN-elicited swallowing was defined as the time between the onset of SLN stimulation and the peak of the VNA. The peak time in the optical signal change was defined as the time from the onset of SLN stimulation to the maximal fluorescence intensity. The decay time was defined as the time from the peak to the point at which ΔF/F returned to the baseline level. All statistical analyses were performed using paired two-tailed Student’s t-tests in GraphPad Prism 8. All statistical data are reported as the mean ± standard error. Significance was set at P < 0.05. Result SLN-evoked bursting of VNA motor discharge reflected the motor pattern that was previously defined as pharyngeal swallow in the arterially perfused brainstem preparation [ 20 ]. In the present study we used SLN stimulations around the pre-determined threshold intensity for evoked swallowing (see Methods). In our experimental setting SLN stimulations randomly failed or successfully triggered VNA swallowing motor discharge (Figs. 2 and 3 ) in n = 8 in situ preparations. SLN-evoked swallowing motor bursting had an average latency of 0.304 ± 0.097s. SLN-evoked swallowing also evoked a phase reset of the respiratory cycle that was characterized by a prolongation of the respiratory cycle of 0.36 ± 0.41 s and indicates appropriate coordination of breathing and swallowing within distributed ponto-medullary motor networks [ 27 ]. We performed Calcium imaging in the dorsal medulla and analyzed the fractional changes in fluorescence intensity ( ΔF/F ) in response to SLN-evoked swallowing. Figure 3 A illustrates Calcium imaging results of pass-fail experiments for SLN-evoked swallowing from a single experiment. In case failure to trigger swallowing motor activity in the VNA recording we observed a spatially confined fluorescent change within the dorsal medulla oblongata (Fig. 3 A). Contrary SLN stimulation that evoked swallowing motor bursts in the VNA were associated with larger spatially distributed Ca 2+ signal across major segments of the dorsal rostro-caudal medulla oblongata (Fig. 3 B). Group data indicate that mean change in fluorescence intensity ( ΔF/F) was significantly lower (p = 0.042) in experiments where SLN-stimulation failed to elicit swallowing (1.00 ± 0.46%) compared to the ΔF/F observed after a SLN- evoked swallowing burst in the VNA (1.53 ± 0.85%). In addition, the latency for the peak Ca 2+ signal was shorter for SLN-stimulation that failed to evoke swallowing motor activity (114.3 ± 94.4 ms), compared to the latency of peak ΔF/F observed after SLN-evoked swallowing (200.0 ± 145.2 ms). The difference between the two latencies was not statistically significant (p = 0.070, see discussion). However, the latencies for the onset of Ca2 + and the decay time of the fluorescent signal were increased when SLN stimulation evoked swallowing compared to SLN stimulations that failed to trigger a swallow (514.3 ± 382.6 ms vs. 314.3 ± 100.8 ms; p = 0.286). Anatomical verification of the source of the fluorescent signals (Fig. 4 ) revealed that, the region activated by electrical stimulation of the SLN was located within the boundaries of the NTS, which can be easily identified via the 4th ventricle and calamus scriptorius indicated by the dot (Calamus) dotted lines (ventricle) in Fig. 4 . Importantly the Ca 2+ fluorescence signals associated with SLN-stimulations that failed to evoke a swallowing response were exclusively localized on the ipsilaterally within the NTS. Contrary SLN-stimulations that evoked the swallowing motor pattern in VNA recordings were associated with widely distributed Ca 2+ signals across the bilateral surface of the dorsal medulla oblongata. The latter clearly indicate the activation of the extended swallowing premotor network of the DSG. (Fig. 4 c). Discussion In the present study, we conducted optical recording and obtained Ca 2+ signals from the dorsal surface of the medulla using an in situ perfused brainstem preparation after stimulation of the SLN. The present study illustrates the spatio-temporal network response of the relay of afferent oropharyngeal sensory input the activity of the dorsal swallowing group (DSG) within the anatomical boundaries of the nucleus of the solitary tract. The spatial resolution of the optical response of the synaptic relay of afferent signals from the SLN. The terminal fields of afferent input from the SLN were anatomically identified to be predominantly located in the ipsilateral interstitial subnuclei of the NTS [ 28 , 29 ]. The SLN relay neurons neurons receive oligosynaptic inputs from the SLN afferent mediate the initiation of the swallowing motor sequence [ 16 ]. The present imaging study shows the spatio-temporal separation between the ipsilaterally neuronal activation of sensory relay neurons within discrete subregions of the NTS and SIN activation within the DSG with two lines of evidence. First, the latency for the peak Ca 2+ for SLN stimulation that failed to trigger swallowing motor activity was significantly shorter compared to the response of SLN-evoked swallowing. Second, the Ca 2+ signal that does not accompany swallowing was restricted ipsilaterally and was significantly shorter compared to bilaterally distributed signal originating from SIN activation in the DSG. The present data are in basic agreement with a recent report showing the anatomical separation of sensory relay neurons and SINs of the DSG using GCaMP6f-related calcium imaging approaches at single cell resolution in the perfused brainstem preparation [ 30 ]. However, the latter study identified a scattered distribution of sensory relay neurons across the surface of the entire contralateral ponto-medullary brainstem while the localization of SINs was restricted to areas in and around the NTS [ 30 ]. The discrepancy regarding location of sensory relay neurons may arise from the fact that Koyama and colleagues imaged the brainstem contralateral to the SLN stimulation and thus reported neurons which also may receive second order synaptic inputs for the SLN rather than reflecting the primary sensory relay neurons for SLN-mediated input. The spatiotemporal resolution of the optical signals during SLN-evoked swallowing within the dorsal swallowing group. Previous studies have demonstrated that swallowing interneurons (SINs) are organized in a dorsal and ventral swallowing group in the caudal brainstem [ 7 , 15 ]. SINs of dorsal swallowing group (DSG) have been proposed to be involved in swallowing pattern generation [ 3 ]. Most SINs of the DSG are found in the nucleus tractus solitarius (NTS) and adjacent reticular formation [ 4 , 16 ] and local inhibition of the DSG neurons with a GABA-receptor agonist does indeed abolish sensory evoked swallowing motor activity [ 18 ]. The spatial signature of bilaterally rostrocaudally disrupted Ca 2+ signals match the anatomical locations of identified SINs of DSG in the perfused brainstem preparation [ 4 , 20 , 31 ] and in vivo [ 6 , 10 ]. However, the difference between the latencies of the Ca 2+ signal of the DSG and the swallowing burst in vagal nerve recording (see results) suggests that swallowing network activity in the DSG is further processed elsewhere before the final swallowing motor act. As suggested previously swallowing premotor neuron populations are likely to be located within the ventral swallowing group (VSG [ 3 ]). Recent discoveries show that neurons of the VSG may be intermingeld with the ventral respiratory column [ 17 ] including respiratory key nodes such as postinspiratory complex [ 32 , 33 ] and the pre-Botzinger complex [ 34 ]. The putative interactions of respiratory neurons of the ventral respiratory colum and the VSG could also play a role in the coordination of swallowing and breathing motor activities. Clinical implications Swallowing disorders are prevalent in patients with neurodegenerative [ 35 ] and neurodevelopmental diseases [ 36 ] and in the elderly [ 37 ]. However, there is ongoing debate whether swallowing disorders are linked to the central circuit dysfunction [ 38 ] or due to impaired oropharyngeal sensing [ 39 , 40 ]. The use of the experimental approach of calcium imaging of close threshold stimulation of the SLN in transgene animal models could help to further elucidate the specific effects of imparied sensory gating or circuit dysfunction. Technical considerations In the present study only OGB-1 was used to stain the dorsal medulla oblongata. Interneurons that are responsive to orthodromic SLN stimulation are located more dorsally than nonresponsive neurons [ 20 ]. Since both sensory relay neurons and interneurons were orthodromically stimulated by through the SLN, the difference in peak times was not significant. Although, clearly shorter in cases of SLN stimulation that failed to evoke swallowing. Declarations Author Contribution S.F. and D.M. wrote the main manuscript text ,and S.F. and Y.S. prepared figures. All authors reviewed the manuscript. GRANTS and Conflict of Interests This work was supported by a Grant-in-Aid for Scientific Research (C) (Grant Number 22K09671). The authors have no conflict of interest to declare. References Amri M, Car A (1988) Projections from the medullary swallowing center to the hypoglossal motor nucleus: a neuroanatomical and electrophysiological study in sheep. 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Cite Share Download PDF Status: Published Journal Publication published 25 Jan, 2025 Read the published version in Pflügers Archiv - European Journal of Physiology → Version 1 posted Editorial decision: Revision requested 20 Nov, 2024 Reviews received at journal 15 Nov, 2024 Reviews received at journal 06 Nov, 2024 Reviewers agreed at journal 30 Oct, 2024 Reviewers agreed at journal 27 Oct, 2024 Reviewers agreed at journal 30 Sep, 2024 Reviewers invited by journal 25 Sep, 2024 Editor assigned by journal 20 Sep, 2024 Submission checks completed at journal 20 Sep, 2024 First submitted to journal 17 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5104317","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":380562497,"identity":"54b831c4-9f46-4322-b682-e70bf7b36373","order_by":0,"name":"Shinya Fuse","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBAC9gYg8eEAiMmGEDXAp4UHqJpxxgEGCdK0MPOga8ELeCTSHz62OWNTx8/AlvaYh2EbA3/7AYbiArxacoyNc26kSUg2sB035mG4zSBxJoHBeAYeLfYSOWzSOR8OSxgcYG+T5v13m4HhBgMDUC9ehz3/bQHTArJFnrCWBDNmhhsgLWzHwFoMCGrheWMs2XMmTXJmM1u64RyG2zyGZxIb8PqFhz394Ycfx2z4+dnbzB68YbgtJ3f88DFjfCGGAMyQiAE6ibHNmCgdDEhxyfyYWC2jYBSMglEwIgAAqkdCWbdvvhgAAAAASUVORK5CYII=","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Fuse","suffix":""},{"id":380562498,"identity":"c149573b-a498-4bda-9ee5-cfacaa3ba25f","order_by":1,"name":"Yoichiro Sugiyama","email":"","orcid":"","institution":"Saga University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoichiro","middleName":"","lastName":"Sugiyama","suffix":""},{"id":380562499,"identity":"8cd52855-13d2-4fbd-8b8d-7ba149ac6ea5","order_by":2,"name":"Rishi R. Dhingra","email":"","orcid":"","institution":"Case Western Reserve University","correspondingAuthor":false,"prefix":"","firstName":"Rishi","middleName":"R.","lastName":"Dhingra","suffix":""},{"id":380562500,"identity":"fd98011f-87b0-4a0c-a64e-f9140845e5a8","order_by":3,"name":"Shigeru Hirano","email":"","orcid":"","institution":"Kyoto Prefectural University of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shigeru","middleName":"","lastName":"Hirano","suffix":""},{"id":380562501,"identity":"7ff60d45-17e0-49b1-a0d5-0ef81689b66f","order_by":4,"name":"Mathias Dutschmann","email":"","orcid":"","institution":"Case Western Reserve University","correspondingAuthor":false,"prefix":"","firstName":"Mathias","middleName":"","lastName":"Dutschmann","suffix":""},{"id":380562502,"identity":"f34e089b-0102-43a4-af2c-195d92368de6","order_by":5,"name":"Yasumasa Okada","email":"","orcid":"","institution":"Murayama Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yasumasa","middleName":"","lastName":"Okada","suffix":""},{"id":380562503,"identity":"d4fb0072-d3ce-4f9e-835b-861e0cdf29da","order_by":6,"name":"Yoshitaka Oku","email":"","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yoshitaka","middleName":"","lastName":"Oku","suffix":""}],"badges":[],"createdAt":"2024-09-17 15:12:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5104317/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5104317/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00424-025-03065-9","type":"published","date":"2025-01-25T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":72333758,"identity":"a3818eb3-07cc-4813-b285-d1046cf66016","added_by":"auto","created_at":"2024-12-25 15:25:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1512889,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of the experimental approach for Calcium imaging of swallowing network activity in the dorsal medulla oblongata of arterially perfused brainstem preparations. The preparation was placed in the recording chamber and fixed with ear bars and perfused with carbonated aCSF via a double lumen catheter inserted into the descending aorta. Blood or tissue clots in the perfusate were filtered, and the air bubbles were removed by bubble traps. To monitor ongoing respiratory and evoked swallowing activity phrenic (PNA) and vagal nerve activity (VNA) was recorded using suction electrodes. The distal end of the SLN was connected to the suction electrode for electrical stimulation and peripheral chemoreceptors of the carotid body were stimulated by injecting NaCN through the bypass line of the catheter. After OGB-1 was microinjected with a glass micropipette into the dorsal surface of the medulla, the preparation with its exposed dorsal brainstem surface was positioned under the microscope.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5104317/v1/359f5c6f34f71a66ddd9a801.jpg"},{"id":72333097,"identity":"f4ba97bb-d32b-4b37-ad27-9b18eafbf591","added_by":"auto","created_at":"2024-12-25 15:17:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1439283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Illustration of integrated averaged and superimposed PNA and VNA (n = 8) during SLN-stimulation that failed triggering a swallowing motor response. \u003cstrong\u003eB\u003c/strong\u003e: Illustration of integrated averaged and superimposed PNA and VNA (n = 8) during SLN-stimulation that triggered a swallowing motor response.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5104317/v1/ca472ef6f816d12ff057ccb3.jpg"},{"id":72333098,"identity":"35fa9316-01d6-48dd-98ea-dc8f57656c4d","added_by":"auto","created_at":"2024-12-25 15:17:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1163160,"visible":true,"origin":"","legend":"\u003cp\u003eThe figure illustrating the dorsal medulla oblongata of the \u003cem\u003ein situ\u003c/em\u003e perfused brainstem preparation is a composite of the light microscope photograph and the fluorescent image related to the local OGB-1 microinjection. \u003cstrong\u003eA: \u003c/strong\u003eoptical recording of the location of the peak CA\u003csup\u003e2+\u003c/sup\u003e signal associated with neuronal activation related to sensory inputs of SLN-stimulation that failed to evoke a swallowing motor response. in dorsal medulla oblongata. The activated area on the dorsal side of the medulla oblongata 325 ms after stimulation along with optical signal waveforms of two regions, which are indicated by open squares, is shown. Signals are expressed as the percentage of changes in fluorescence (\u003cem\u003eΔF/F\u003c/em\u003e). \u003cstrong\u003eB:\u003c/strong\u003e A representative optical recording of swallowing activity elicited by SLN stimulation in the same animal.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5104317/v1/6b83cb137290458c9fa62ebb.jpg"},{"id":72333100,"identity":"3958d8a9-10f3-4ec5-b020-47d3b67dc157","added_by":"auto","created_at":"2024-12-25 15:17:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":979660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e A light photograph of the dorsal brainstem and schematic drawing shows the anatomical structure of the exposed dorsal brainstem. The red dot shows the bottom of the floor of the fourth ventricle. In B and C, each activation map was drawn using the activation threshold of 2 × SD of the pre-low-pass (\u003cem\u003eΔF/F\u003c/em\u003e). The activation map of each animal was merged with a representative normal light photograph. swallowing is classified into the swallowing start (Sw onset), the peak (Sw peak), and the swallowing end (Sw end).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Illustration of superimposed activation maps from n=8 preparations where SLN stimulation did not elicit swallowing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC: \u003c/strong\u003eActivation maps from 8 animals were superimposed in cases where SLN stimulation elicited swallowing and the time sequence of the optical signal changes during swallowing at the start of SLN stimulation and the swallowing network response at a latency of 2350, 2850, 3350 and 3850ms.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5104317/v1/b9e37434ca68a9d16b3826c7.jpg"},{"id":74858434,"identity":"e6a1149b-fe0b-4fdc-ae47-651ff2dde603","added_by":"auto","created_at":"2025-01-27 16:09:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5753151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5104317/v1/acfa786f-0c08-431f-9bee-9e7bc18a7278.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spatio-temporal segregation between sensory relay and swallowing pre-motor population activities by optical imaging in the rat nucleus of the solitary tract.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pharyngeal phase of swallowing is initiated by a swallowing central pattern generator (sw-CPG) which is traditionally divided into a dorsal and ventral swallowing group (DSG, VSG) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Previous studies characterized the activity of swallowing interneurons (SINs) using single unit recordings predominantly within the DSG [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. SINs are activated by pharyngeal or laryngeal afferent signals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the location of the DSG largely overlaps anatomically with the nucleus of the solitary tract, which also serves as the primary sensory relay nucleus for pharyngeal and laryngeal afferents [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The spatio-temporal dynamic of the interplay between the sensory relay and DSG within these circuits of the dorsal medulla oblongata remains to be elucidated.\u003c/p\u003e \u003cp\u003eFluorescence calcium imaging approaches at the population level in slices and en bloc brainstem spinal cord preparation was previously used to analyze temporal and spatial changes in respiratory network activity at either the population or the single cell level [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, investigation of the network mechanism that underlies the generation of the swallowing motor pattern in relation to breathing requires \u003cem\u003ein vivo\u003c/em\u003e experimental models [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] or alternative approaches that can maintain network function in an \u003cem\u003ein vitro\u003c/em\u003e slice preparation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] or in \u003cem\u003ein situ\u003c/em\u003e arterially perfused brainstem preparations. The latter has become an established experimental model to study the coordination of breathing and swallowing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The perfused brainstem preparation was previously used to image respiratory [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and swallowing activities [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] in brainstem circuits and this specific preparation provides several advantages compared to \u003cem\u003ein vivo\u003c/em\u003e or \u003cem\u003ein vitro\u003c/em\u003e approaches [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The overall mechanical stability in the absence of cardiac pulsation or respiratory movements and the full exposure of the dorsal surface of the brainstem provide a viable experimental approach for calcium imaging and allows for the study of spatio-temporal dynamics of network activity of the DSG and the sensory relay neurons in the dorsal medulla oblongata.\u003c/p\u003e \u003cp\u003eIn the present study we demonstrate that activation of the sensory inputs triggered a spatially confined population Ca\u003csup\u003e2+\u003c/sup\u003e signal within the NTS at short latency after electrical stimulation of the SLN. In contrast, the generation of the swallowing motor pattern occurred after significantly longer latency and engaged DSG population activities along the rostro-caudal and medio-lateral dorsal medulla. The data of the present study demonstrates for the first time that the sensory relay neurons in the NTS spatially overlap with the DSG but are functionally and anatomically separable. In addition, the present study further supports the hypothesis that swallowing motor activity is generated in the DSG and pre-motor populations in the ventral swallowing group transmit the swallowing network activity to the motor pools [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] since the Ca\u003csup\u003e2+\u003c/sup\u003e signals of the DSG clearly preceded the onset of swallowing motor burst.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e The experimental procedures were carried out in accordance with the principles for the Care and Use of Animals of the Physiological Society in Japan and approved by the local University Committee for the Use of Animals in Research, and all experiments were performed at the Hyogo College of Medicine.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003earterially perfused-brainstem preparation.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExperiments were performed using arterially perfused brainstem preparations as previously described in detail [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A total of eight Sprague-Dawley rats (either sex; postnatal days 15\u0026ndash;24; weight: 40.1\u0026ndash;43.8 g) were used. Each animal was initially anesthetized with isoflurane. The left phrenic nerve was isolated and cut just above the diaphragm. In the neck, the left vagus nerve and left SLN were isolated and cut distally. The cerebellum was removed by cutting the cerebellar brachium, and the dorsal medulla oblongata was exposed. Artificial cerebrospinal fluid (aCSF; in mM: 125 NaCl, 3 KCl, 1.25 KH2PO4, 2.5 CaCl2, 1.25 MgSO4, 25 NaHCO3, and 10 D-glucose) containing 4.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e g/mL sucrose was perfused through a catheter using a peristaltic pump (Watson and Marlow, 520s). The perfusate was then bubbled continuously with carbogen (95% O2 and 5% CO2). The flow rate was gradually increased to 16\u0026ndash;22 ml/min so that the characteristic eupneic post-inspiratory motor discharge was established in the vagal nerve recording [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Neuromuscular paralysis was induced by the addition of vecuronium bromide (initial injection of 1.5 mg/kg, additional injections of 0.75 mg/kg when necessary) to the perfusate which suppressed respiratory-related movements of the preparation. To provide a powerful transient excitatory drive for the respiratory brainstem network during the tuning phase of the preparation peripheral chemoreceptors were occasionally stimulated by injecting sodium cyanide (NaCN; 0.1\u0026ndash;0.2 ml, 0.1%, w/v in saline) into the perfusion circuit.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNerve recording\u003c/h2\u003e \u003cp\u003eIn all experiments phrenic nerve activity (PNA) and vagal nerve activity (VNA) were recorded via suction electrodes. The activity of neurons was amplified (AB651J, Nihon Kohden, Tokyo, Japan), bandpass filtered from 15 Hz to 3 kHz, sampled at 800 Hz, and stored together with imaging data using an optical recording system (MiCAM Ultima, BrainVision, Tokyo, Japan). The nerve discharges were high pass filtered (cutoff frequency\u0026thinsp;=\u0026thinsp;0.1 Hz), fully rectified and leaky integrated at a time constant of 20 ms.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental protocol for SLN stimulation\u003c/h3\u003e\n\u003cp\u003eFor electrical stimulation of the SLN a bipolar electrode was used to apply short trains of electrical pulses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] with a duration of 0.2 ms at a frequency of 10\u0026ndash;20 Hz and at an intensity of (20\u0026ndash;50 \u0026micro;A). Initially we determined the threshold of the stimulus intensity for SLN-evoked swallows which can be reliably identified in vagal nerve recordings [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Subsequently we performed SLN-stimulations at the threshold level during the optical recording sessions (details below). This allowed us to discriminate the optical signals only related to activation of the sensory relay neurons in the NTS in case SLN-stimulation failed to evoke swallowing. SLN-stimulations that succeeded to evoke swallowing motor activity then revealed spatio-temporal network activity of the overlapping DSG.\u003c/p\u003e\n\u003ch3\u003eCalcium-sensitive dye microinjection\u003c/h3\u003e\n\u003cp\u003eWe stained the dorsal medulla with the calcium indicator Oregon Green 488 BAPTA-1 acetoxymethyl (OGB-1, Invitrogen, Carlsbad, CA, USA). OGB-1 was dissolved in 10% Pluronic F-127 in dimethyl sulfoxide (DMSO, Invitrogen, Carlsbad, CA, USA) at a concentration of 200 \u0026micro;M. The microinjection sites were located 0.5\u0026ndash;1.0 mm rostral to the obex; 0.5\u0026ndash;1.0 mm lateral from the midline on both sides; and 300, 400, 500 \u0026micro;m below the dorsal surface. A total of 50\u0026ndash;60 nl of OGB-1 was microinjected into each injection site.\u003c/p\u003e\n\u003ch3\u003eOptical recording\u003c/h3\u003e\n\u003cp\u003eAfter dye microinjection, the recording chamber was carefully placed under a macro zoom fluorescence microscope (MVX-10, Olympus Optical, Tokyo, Japan) using a magnification of 1.6 x. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Time-lapsed Ca\u003csup\u003e2+\u003c/sup\u003e signals in the dorsal medulla were imaged using an optical recording system (MiCAM Ultima, BrainVision, Tokyo, Japan). Preparations were illuminated with a tungsten-halogen lamp (150 W) through a bandpass excitation filter (λ\u0026thinsp;=\u0026thinsp;460\u0026ndash;495 nm). Epifluorescence was detected through a long-pass barrier filter (λ\u0026thinsp;\u0026gt;\u0026thinsp;510 nm) with a CMOS sensor array (MiCAM Ultima L-camera, BrainVision; 100 \u0026micro;m \u0026times; 100 \u0026micro;m pixel size, 100 \u0026times; 100 pixel array). Optical signals were sampled at 40 Hz (25 ms/frame). A total of 256 frames were recorded starting at 64 frames (1.6 s) before the onset of SLN electrical stimulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eImage processing\u003c/h3\u003e\n\u003cp\u003eFirst, the change in fluorescence intensity (\u003cem\u003eΔF\u003c/em\u003e) relative to the initial intensity (\u003cem\u003eF0\u003c/em\u003e) was calculated. Then, the fractional change in fluorescence intensity (\u003cem\u003eΔF/F\u003c/em\u003e) at each pixel in each frame was calculated based on the background fluorescence intensity (F). Subsequently, the images were smoothed spatially using a 5 \u0026times; 5 pixel spatial filter; pre-low-pass image) and temporally using a low-pass filter (cutoff frequency\u0026thinsp;=\u0026thinsp;2 Hz; low-pass image).\u003c/p\u003e \u003cp\u003eNext, we selected 9\u0026ndash;20 images from each animal in which SLN stimulation did not elicit swallowing and applied cycle-triggered averaging. For SLN stimulation that elicited swallowing we selected 5\u0026ndash;20 images from each animal and also applied cycle-triggered averaging. Finally, the activation map of the low-pass image was drawn using an activation threshold of 2 \u0026times; SD (standard deviation). The SD was derived from \u003cem\u003eΔF/F\u003c/em\u003e at each pixel within 10 frames before the onset of SLN stimulation in the pre-low-pass image. The activation map for each animal was superimposed based on the bottom of the floor of the fourth ventricle of each animal and merged with a representative normal light-microscope photograph.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eWe measured the latency to SLN-elicited swallowing activity at the neurogram and measured the peak and decay times of optical signal changes. The latency of SLN-elicited swallowing was defined as the time between the onset of SLN stimulation and the peak of the VNA. The peak time in the optical signal change was defined as the time from the onset of SLN stimulation to the maximal fluorescence intensity. The decay time was defined as the time from the peak to the point at which \u003cem\u003eΔF/F\u003c/em\u003e returned to the baseline level.\u003c/p\u003e \u003cp\u003eAll statistical analyses were performed using paired two-tailed Student\u0026rsquo;s t-tests in GraphPad Prism 8. All statistical data are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003eSLN-evoked bursting of VNA motor discharge reflected the motor pattern that was previously defined as pharyngeal swallow in the arterially perfused brainstem preparation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present study we used SLN stimulations around the pre-determined threshold intensity for evoked swallowing (see Methods). In our experimental setting SLN stimulations randomly failed or successfully triggered VNA swallowing motor discharge (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) in n\u0026thinsp;=\u0026thinsp;8 \u003cem\u003ein situ\u003c/em\u003e preparations. SLN-evoked swallowing motor bursting had an average latency of 0.304\u0026thinsp;\u0026plusmn;\u0026thinsp;0.097s. SLN-evoked swallowing also evoked a phase reset of the respiratory cycle that was characterized by a prolongation of the respiratory cycle of 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 s and indicates appropriate coordination of breathing and swallowing within distributed ponto-medullary motor networks [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe performed Calcium imaging in the dorsal medulla and analyzed the fractional changes in fluorescence intensity (\u003cem\u003eΔF/F\u003c/em\u003e) in response to SLN-evoked swallowing. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA illustrates Calcium imaging results of pass-fail experiments for SLN-evoked swallowing from a single experiment. In case failure to trigger swallowing motor activity in the VNA recording we observed a spatially confined fluorescent change within the dorsal medulla oblongata (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Contrary SLN stimulation that evoked swallowing motor bursts in the VNA were associated with larger spatially distributed Ca\u003csup\u003e2+\u003c/sup\u003e signal across major segments of the dorsal rostro-caudal medulla oblongata (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Group data indicate that mean change in fluorescence intensity (\u003cem\u003eΔF/F)\u003c/em\u003e was significantly lower (p\u0026thinsp;=\u0026thinsp;0.042) in experiments where SLN-stimulation failed to elicit swallowing (1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46%) compared to the \u003cem\u003eΔF/F\u003c/em\u003e observed after a SLN- evoked swallowing burst in the VNA (1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85%). In addition, the latency for the peak Ca\u003csup\u003e2+\u003c/sup\u003e signal was shorter for SLN-stimulation that failed to evoke swallowing motor activity (114.3\u0026thinsp;\u0026plusmn;\u0026thinsp;94.4 ms), compared to the latency of peak \u003cem\u003eΔF/F\u003c/em\u003e observed after SLN-evoked swallowing (200.0\u0026thinsp;\u0026plusmn;\u0026thinsp;145.2 ms). The difference between the two latencies was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.070, see discussion). However, the latencies for the onset of Ca2\u0026thinsp;+\u0026thinsp;and the decay time of the fluorescent signal were increased when SLN stimulation evoked swallowing compared to SLN stimulations that failed to trigger a swallow (514.3\u0026thinsp;\u0026plusmn;\u0026thinsp;382.6 ms vs. 314.3\u0026thinsp;\u0026plusmn;\u0026thinsp;100.8 ms; p\u0026thinsp;=\u0026thinsp;0.286).\u003c/p\u003e \u003cp\u003eAnatomical verification of the source of the fluorescent signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed that, the region activated by electrical stimulation of the SLN was located within the boundaries of the NTS, which can be easily identified via the 4th ventricle and calamus scriptorius indicated by the dot (Calamus) dotted lines (ventricle) in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Importantly the Ca\u003csup\u003e2+\u003c/sup\u003e fluorescence signals associated with SLN-stimulations that failed to evoke a swallowing response were exclusively localized on the ipsilaterally within the NTS. Contrary SLN-stimulations that evoked the swallowing motor pattern in VNA recordings were associated with widely distributed Ca\u003csup\u003e2+\u003c/sup\u003e signals across the bilateral surface of the dorsal medulla oblongata. The latter clearly indicate the activation of the extended swallowing premotor network of the DSG. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we conducted optical recording and obtained Ca\u003csup\u003e2+\u003c/sup\u003e signals from the dorsal surface of the medulla using an \u003cem\u003ein situ\u003c/em\u003e perfused brainstem preparation after stimulation of the SLN. The present study illustrates the spatio-temporal network response of the relay of afferent oropharyngeal sensory input the activity of the dorsal swallowing group (DSG) within the anatomical boundaries of the nucleus of the solitary tract.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe spatial resolution of the optical response of the synaptic relay of afferent signals from the SLN.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe terminal fields of afferent input from the SLN were anatomically identified to be predominantly located in the ipsilateral interstitial subnuclei of the NTS [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The SLN relay neurons neurons receive oligosynaptic inputs from the SLN afferent mediate the initiation of the swallowing motor sequence [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present imaging study shows the spatio-temporal separation between the ipsilaterally neuronal activation of sensory relay neurons within discrete subregions of the NTS and SIN activation within the DSG with two lines of evidence. First, the latency for the peak Ca\u003csup\u003e2+\u003c/sup\u003e for SLN stimulation that failed to trigger swallowing motor activity was significantly shorter compared to the response of SLN-evoked swallowing. Second, the Ca\u003csup\u003e2+\u003c/sup\u003e signal that does not accompany swallowing was restricted ipsilaterally and was significantly shorter compared to bilaterally distributed signal originating from SIN activation in the DSG. The present data are in basic agreement with a recent report showing the anatomical separation of sensory relay neurons and SINs of the DSG using GCaMP6f-related calcium imaging approaches at single cell resolution in the perfused brainstem preparation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, the latter study identified a scattered distribution of sensory relay neurons across the surface of the entire contralateral ponto-medullary brainstem while the localization of SINs was restricted to areas in and around the NTS [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The discrepancy regarding location of sensory relay neurons may arise from the fact that Koyama and colleagues imaged the brainstem contralateral to the SLN stimulation and thus reported neurons which also may receive second order synaptic inputs for the SLN rather than reflecting the primary sensory relay neurons for SLN-mediated input.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe spatiotemporal resolution of the optical signals during SLN-evoked swallowing within the dorsal swallowing group.\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that swallowing interneurons (SINs) are organized in a dorsal and ventral swallowing group in the caudal brainstem [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. SINs of dorsal swallowing group (DSG) have been proposed to be involved in swallowing pattern generation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Most SINs of the DSG are found in the nucleus tractus solitarius (NTS) and adjacent reticular formation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and local inhibition of the DSG neurons with a GABA-receptor agonist does indeed abolish sensory evoked swallowing motor activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The spatial signature of bilaterally rostrocaudally disrupted Ca\u003csup\u003e2+\u003c/sup\u003e signals match the anatomical locations of identified SINs of DSG in the perfused brainstem preparation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the difference between the latencies of the Ca\u003csup\u003e2+\u003c/sup\u003e signal of the DSG and the swallowing burst in vagal nerve recording (see results) suggests that swallowing network activity in the DSG is further processed elsewhere before the final swallowing motor act. As suggested previously swallowing premotor neuron populations are likely to be located within the ventral swallowing group (VSG [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]). Recent discoveries show that neurons of the VSG may be intermingeld with the ventral respiratory column [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] including respiratory key nodes such as postinspiratory complex [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and the pre-Botzinger complex [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The putative interactions of respiratory neurons of the ventral respiratory colum and the VSG could also play a role in the coordination of swallowing and breathing motor activities.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eClinical implications\u003c/h2\u003e \u003cp\u003eSwallowing disorders are prevalent in patients with neurodegenerative [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and neurodevelopmental diseases [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and in the elderly [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, there is ongoing debate whether swallowing disorders are linked to the central circuit dysfunction [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] or due to impaired oropharyngeal sensing [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The use of the experimental approach of calcium imaging of close threshold stimulation of the SLN in transgene animal models could help to further elucidate the specific effects of imparied sensory gating or circuit dysfunction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTechnical considerations\u003c/h2\u003e \u003cp\u003eIn the present study only OGB-1 was used to stain the dorsal medulla oblongata. Interneurons that are responsive to orthodromic SLN stimulation are located more dorsally than nonresponsive neurons [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Since both sensory relay neurons and interneurons were orthodromically stimulated by through the SLN, the difference in peak times was not significant. Although, clearly shorter in cases of SLN stimulation that failed to evoke swallowing.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.F. and D.M. wrote the main manuscript text ,and S.F. and Y.S. prepared figures. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch3\u003eGRANTS and Conflict of Interests\u003c/h3\u003e\n\u003cp\u003eThis work was supported by a Grant-in-Aid for Scientific Research (C) (Grant Number 22K09671). The authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmri M, Car A (1988) Projections from the medullary swallowing center to the hypoglossal motor nucleus: a neuroanatomical and electrophysiological study in sheep. Brain Res. 441:119-126. https://doi.org/10.1016/0006-8993(88)91389-3\u003c/li\u003e\n\u003cli\u003eEzure K, Oku Y, Tanaka I (1993) Location and axonal projection of one type of swallowing interneurons in cat medulla. Brain Res. 632:216-224. https://doi.org/10.1016/0006-8993(93)91156-M\u003c/li\u003e\n\u003cli\u003eJean A (2001) Brain stem control of swallowing: neuronal network and cellular mechanisms. Physiol Rev. 81(2): 929-969. https://doi.org/10.1152/physrev.2001.81.2.929\u003c/li\u003e\n\u003cli\u003eHashimoto K, Sugiyama Y, Fuse S, Umezaki T, Oku Y, Dutschmann M, Hirano S (2018) Activity of Swallowing-Related Neurons in the Medulla in the Perfused Brainstem Preparation in Rats. 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Dysphagia. 14 (2): 93-109. https://doi.org/10.1007/PL00009593\u003c/li\u003e\n\u003cli\u003eAlvarez-Berdugo D, Rofes L, Casamitjana JF, Padr\u0026oacute;n A, Quer M, Clav\u0026eacute; P (2016) Oropharyngeal and laryngeal sensory innervation in the pathophysiology of swallowing disorders and sensory stimulation treatments. Ann N Y Acad Sci. 1380 (1): 104-120. https://doi.org/10.1111/nyas.13150\u003c/li\u003e\n\u003cli\u003eSantoso LF, Kim DY, Paydarfar D (2019) Sensory dysphagia: A case series and proposed classification of an under recognized swallowing disorder. Head Neck 41 (5): E71-E78. https://doi.org/10.1002/hed.25588\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Swallowing, In situ perfused brainstem preparation, Optical recording, Calcium imaging, Dorsal swallowing group","lastPublishedDoi":"10.21203/rs.3.rs-5104317/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5104317/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nucleus tractus solitarius (NTS) contains neurons that relay sensory swallowing commands information from the oropharyngeal cavity and swallowing premotor neurons of the dorsal swallowing group (DSG). However, the spatio-temporal dynamics of the interplay between the sensory relay and the DSG is not well understood. Here we employed fluorescence imaging after microinjection of the calcium indicator into the NTS in an arterially perfused brainstem preparation of rat (n\u0026thinsp;=\u0026thinsp;8) to investigate neuronal population activity in the NTS in response to superior laryngeal nerve (SLN) stimulation. Respiratory and swallowing motor activities were determined by simultaneous recordings of phrenic and vagal nerve activity (PNA, VNA). Analysis of SLN stimulation near the threshold triggering a swallowing allowed us to analyze Ca\u003csup\u003e2+\u003c/sup\u003e signals related to the sensory relay and the DSG. We show that activation of sensory relay neurons triggers spatially confined Ca\u003csup\u003e2+\u003c/sup\u003e signals exclusively unilateral to the stimulated SLN at short latencies (114.3\u0026thinsp;\u0026plusmn;\u0026thinsp;94.4 ms). However, SLN-evoked swallowing triggered Ca\u003csup\u003e2+\u003c/sup\u003e signals bilaterally at longer latencies (200\u0026thinsp;\u0026plusmn;\u0026thinsp;145.2 ms) and engaged anatomically distributed DSG activity across the dorsal medulla oblongata. The Ca\u003csup\u003e2+\u003c/sup\u003e signals originating from the DSG preceded evoked VNA swallow motor bursts, thus the swallowing premotor neurons that drive laryngeal motor pools are located outside the DSG. In conclusion, the study illuminates the spatial-temporal features of sensory-motor integration of swallowing in the NTS and further supports the hypothesis that the NTS harbors swallowing pre-motor neurons that may generate the swallowing motor activity while first order pre-motor pools are located outside the DSG.\u003c/p\u003e","manuscriptTitle":"Spatio-temporal segregation between sensory relay and swallowing pre-motor population activities by optical imaging in the rat nucleus of the solitary tract.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-25 15:17:45","doi":"10.21203/rs.3.rs-5104317/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-20T15:58:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-16T02:44:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-07T00:09:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15378391629594759897679637365164738901","date":"2024-10-30T16:21:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48602134885039835463992380080186987307","date":"2024-10-27T23:19:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240651456381145873629302578601414032355","date":"2024-09-30T15:16:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-25T06:36:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-20T09:33:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-20T09:29:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pflügers Archiv - European Journal of Physiology","date":"2024-09-17T15:11:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pflugers-archiv-european-journal-of-physiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paej","sideBox":"Learn more about [Pflügers Archiv - European Journal of Physiology](http://link.springer.com/journal/424)","snPcode":"424","submissionUrl":"https://submission.nature.com/new-submission/424/3","title":"Pflügers Archiv - European Journal of Physiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02c219f6-d0de-4f57-a563-c418aa6f8f94","owner":[],"postedDate":"December 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-27T16:02:32+00:00","versionOfRecord":{"articleIdentity":"rs-5104317","link":"https://doi.org/10.1007/s00424-025-03065-9","journal":{"identity":"pflugers-archiv-european-journal-of-physiology","isVorOnly":false,"title":"Pflügers Archiv - European Journal of Physiology"},"publishedOn":"2025-01-25 15:57:43","publishedOnDateReadable":"January 25th, 2025"},"versionCreatedAt":"2024-12-25 15:17:45","video":"","vorDoi":"10.1007/s00424-025-03065-9","vorDoiUrl":"https://doi.org/10.1007/s00424-025-03065-9","workflowStages":[]},"version":"v1","identity":"rs-5104317","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5104317","identity":"rs-5104317","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}